Recently, there has been a substantial increase in the incidence of human papillomavirus (HPV)-associated oropharyngeal (OP) squamous cell carcinoma (OPSCC), while other cases result from alcohol and/or tobacco use. Since HPV infection confers a significantly decreased mortality rate, understanding the molecular mechanisms responsible for HPV-associated oral carcinoma is crucial to understanding the pathways regulated by HPV versus those involved in HPV-negative oral cancer. Recently, a subpopulation of HPV-positive OPSCC patients with tobacco exposure and p53 genetic alterations has been identified, warranting detailed investigation of the HPV E6- and p53-mediated pathways contributing to carcinogenesis. In HPV-associated OPSCC, the viral E6 and E7 oncoproteins are expressed at high levels. MicroRNAs (miRNAs) are ~22 nt long single-stranded RNAs that generally function as negative regulators of gene expression, and their expression profiles are altered in a variety of human cancers. Limited information is available on the role of miRNAs in the pathogenesis of OP cancers. Our published studies using OPSCC cell lines show that miRNA expression profiles in HPV-positive and HPV-negative OP cancers are distinctively different from each other, including the overexpression of miR-363 and underexpression of miR-218 in HPV-positive cell lines. MiR-363 is also overexpressed in HPV-16 positive OP cancer tissues compared to HPV-negative OPSCC tissues, normal oral mucosa and normal oral keratinocytes (NOKs). Our data also show that the HPV-16 E6 oncogene promotes miR-363 overexpression while reducing miR-218 expression. We hypothesize that differences in miR-363 and/or miR-218 levels change the expression of their target genes and this alters the cellular pathways involved in HPV-positive oral carcinogenesis. In Aim 1, we will test whether miR-363 and miR- 218 are oncogenic and tumor suppressor miRNAs, respectively, that affect important cellular pathways. We will accomplish this by overexpressing or knocking-down the expression of miR-363 and miR-218 in HPV-positive and HPV-negative OPSCC cell lines, NOKs and NOK expressing E6, and study their effect on cell proliferation, migration, invasion, adhesion and apoptosis. In Aim 2, we will identify the regulatory mechanisms by which E6 affects miR-363 and miR-218 expression and whether these are p53-dependent. We will use inhibitors of DNA methylation and histone modification to test whether epigenetic processes are involved in E6-mediated regulation of these miRNAs. If this is the case, we will also study the methylation status and chromatin state of these miRNA promoters. We will also test whether E6 affects miR-363 and miR-218 expression through inactivation of the p53 pathway. Our studies should provide valuable information on the role of miR-363 and miR-218 in HPV-positive OP cancers, the nature of regulation of these miRNAs by E6, and the molecular basis for the differential regulation of miR-363 and miR-218 in HPV-positive and HPV-negative OPSCC. This information could be utilized for future diagnosis, prognosis and therapeutic targets for these cancers.